Lysostaphin-based assay of human granulocyte functions: a reevaluation

Lysostaphin, a staphylococcus-derived staphylocidal substance, has widely been used in assays of granulocyte phagocytic and bactericidal capability. It rapidly kills extracellular bacteria. Thus, a separate determination of intracellular surviving bacteria can be performed. One prerequisite for this approach is the safe inactivation of lysostaphin (usually brought about by trypsin) before the intracellular bacteria are externalized for plating. This inactivation has been found by others to be incomplete. Data are presented demonstrating a safe inactivation of lysostaphin by trypsin, if the pH value is maintained within the alkaline range. A low variation of results is obtained by plotting the total number of bacteria killed per incubate vs the logarithm of initial bacterial inoculum or of the intracellular surviving bacteria, leading to linear regression lines. The variation of the results increases greatly for initial bacteria/granulocyte proportions of greater than 5/1. The results obtained for two different St. aureus strains are significantly different. Dexamethasone pretreatment (12 mg p.o. within 8 h) had no detectable influence, when fresh blood was assayed, while blood storage at room temperature for 12 h (without dexamethasone pretreatment) led to a significant functional impairment, mainly of bactericidal capability when analyzed in a pairwise fashion. A major limitation of this kind of assays is that killed bacteria cannot be determined directly.